Ca2+ is a second messenger regulating many vital processes; therefore, cellular mechanisms for inciting, terminating, and shaping Ca2+ signals attract intense research. Ca2+ fluxes between the cytosol and both the extracellular space and the endoplasmic reticulum (ER) are the best-understood components of Ca2+ dynamics. However, other organelles, such as secretory granules (SG), may also contribute. It has long been known that SG maintain Ca2+ levels well above those of the cytosol. Recent work demonstrating the presence of functional release channels and emphasizing the highly localized action of Ca2+ signals further argues that SG represent important components of the cellular Ca2+ signaling ma- chinery. A confounding factor has been that SG transport protons as well as Ca2+; this complicates Ca2+ measurements by affecting Ca2+ reporters and altering SG Ca2+ buffering. The research in this proposal will quantitatively dissect global cellular Ca2+ transport in SG-containing cells, characterize the Ca2+ buffering capacity of SG, and measure the response of SG Ca2+ to cell stimuli while measuring pH effects on reporters and SG buffers. Ca2+ and pH photometry using both chemical probes and genetically-encoded reporters will be used in both intact neuroendocrine cells and isolated SG to achieve these goals. The results will lead to better understanding of Ca2+ dynamics in neuroendocrine cells, as well as in other clinically relevant cells in which SG Ca2+ is potentially important, including pancreatic beta-cells and mast cells.